Ake Holmberg and Ernst H. Bdrdny

Transcription

1 The effect of pilocarpine on the endothelium forming the inner wall of Schlemm's canal: An electron-microscopic study in the monkey Cercopithecus aethiops Ake Holmberg and Ernst H. Bdrdny Eight Cercopithecus aethiops monkeys were treated topically in one eye with a close of pilocarpine that gives a large facility increase. The chamber angles were fixed in buffered osmic acid and the endothelial layer forming the inner wall of Schlemm's canal studied with the electron microscope. Vacuole-like structures, which would include sections through transendothelial channels, were counted, as were sections through the nuclei. The frequency of vacuole-like structures, but not of sections through the nuclei, was reduced by nearly half on the treated side. There was no correlation between this lowering and any pilocarpine effect on facility. It is pointed out that the frequency with which a structure is found in ultrathin sections can be decreased because of a true reduction in incidence or because of a reduction in the chance of being included in a section through changes in shape or size. Thus, the functional significance of the present findings is not clear. I t is well known that miotics increase the facility of aqueous humour outflow. However, it is not fully understood how this effect is accomplished. The general opinion From the Eye Department, Sabbatsbergs Hospital, the Department of Anatomy, Karolinska Institute, Stockholm (Dr. Holmberg), the Department of Physiology, Makerere University College Medical School, Kampala, Uganda, and the Department of Pharmacology, University of Uppsala, Sweden (permanent address of Dr. Barany). This study was supported by Research Grants NB (Dr. Barany) and NB (Dr. Holmberg) from the Institute of Neurological Diseases and Blindness, United States Public Health Service, Bethesda, Md., and from the Swedish Medical Research Council (Dr. Holmberg). 53 is that the intertrabecular spaces become wider because of ciliary muscle pull. The possible existence of an additional effect of pilocarpine was suggested by Barany. 1 Because of the slowness with which systemic atropine reversed the action of topical pilocarpine on facility in monkey eyes, he questioned if the facility increasing effect of pilocarpine might not have another component unrelated to the ciliary muscle contraction, an action directly upon channels through the endothelium of the inner wall of Schlemm's canal. These channels have been described by Holmberg 2 ' :t in studies of the inner wall of Schlemm's canal. The wall in a strict sense is built of a single layer of flat endothelial cells. "Vacuoles" were found in these

2 54 Holmberg and Bdrdnij Investigative Ophthalmology February 1966 cells which, by serial sections, were proved to be small channels through the wall. These channels are believed to be the final pathways for the aqueous humor through the trabecular meshwork. Because of their small dimensions, they might collectively constitute the main resistance to outflow. We have, therefore, conducted the following experiments to see if pilocarpine causes these channels to undergo any change that might reduce their collective resistance. Material and methods Eight adult vervet monkeys (Cercopithecus aethiops) were used. In each animal, one eye was the experimental and the other served as control and received saline. A few days before the experiments, both eyes were checked for normality with a slit lamp and ophthalmoscope under phencyclidine. 4 Perfusion and fixation. The monkeys were anesthetized with intravenous pentobarbital (Veterinary Nembutal). One eye was treated with 200 fig pilocarpine hydrochloride. The drug was dissolved as 2 per cent solution in 0.4 per cent saline and 10 v\ of the solution was applied to the cornea and allowed to dry for 2 minutes, similarly to the experiments of Tornqvist. 5 This dose, applied in this manner, regularly causes a maximum facility increase in the treated eye. For safety's sake, however, the facility increases were checked in each eye by the two-level constant pressure perfusion technique. One hour after the dose, both eyes were punctured using the needle gun of Sears 0 and the anterior chamber infused for 4 to 5 minutes first at 2.5 mm. Hg above resting pressure, then at 11.9 mm. Hg above resting pressure, and then, as a rule, again 4 to 5 minutes at the lower pressure. The buffered solution described by Barany 7 was used. The changes in infusion rate were used for estimating facility. 7 The infusion was interrupted by closing of stopcocks and the needle in each eye shot through the opposite side of the cornea. The tubing to the infusion system was then cut and the eyes enucleated with the animal still living and the anterior chamber uncollapsed. The second eye was enucleated only after the first one was in osmic acid. Each eye was treated as quickly as possible after enucleation. Small segments of the anterior part were cut out with razor blades and put into Sjdstrand's chilled, buffered osmic acid s within 60 seconds of enucleation. Once the segment was in osmic acid, the triangular flap of iris in the segment was very gently pulled away from the cornea, so that the fixative could approach the chamber angle region without delay. Treatment of the fixed material. After one hour in osmic acid, the cutout segments were washed in perfusion fluid for about one hour with frequent changes, then transferred to 70 per cent v/v ethanol with one per cent phosphotungstic acid, and then to 70 per cent ethanol. The segments were kept in this solution for several weeks during transport from Africa to Stockholm. They were then transferred to 90 per cent ethanol for one hour and the blocks trimmed to smaller pieces (containing 2 to 4 mm. of the length of Schlemm's canal), and placed into the gelatine capsules used for embedding. Dehydration was then continued in 100 per cent ethanol and the tissue embedded in Epon, following the procedure described by Luft.!) Polymerization was done at 60 C. for about 48 hours. After hardening, the blocks were trimmed with razor blades and the part of the tissue possibly damaged by cutting in the unembedded state was carefully removed. Fig. 1 shows the orientation of the tissue with Fig. 1. Schematic drawing showing orientation of the specimen for sectioning. The area marked with a rectangle in A is magnified in B and indicates the approximate size of that part of Schlemm's canal and the trabecular meshwork, which was used for cutting. I is the plane of sectioning, 2, Schlemm's canal, 3, sclera, 4, trabecular meshwork, 5, anterior chamber, 6, iris, and 7, inner wall of Schlemm's canal.

3 Volume 5 Number L Pilocarpine effect in Schlemm's canal 55 respect to the plane of section. Sectioning was done on an LKB-ultratome with glass knives to a thickness of about 0.05 A. The sections were examined in an Akashi-50 or Hitashi-7H election microscope. Selection of sections. The procedure for choosing sections to be evaluated was as follows: 1. A series of 25 to 50 consecutive sections were cut at one level of the tissue block, then a fraction of a millimeter of the block was removed with razor blades and another series of 25 to 50 sections were cut. In this manner, five series were taken from every eye of the pilocarpine series. Mostly they belonged to the same block and thus were derived from the same neighborhood of the trabecular meshwork. 2. The sections of each series were scanned optically at low magnification on the fluorescent screen of the electron microscope and one section from each series selected for technical perfection exclusively, which means that the whole section was placed accessibly on the grid and that no folds or scratches prevented part of the section from being used. At the low magnification used, only the very largest vacuoles would have been visible anyhow. This selected section was photographed at a primary magnification of x600 to 1, From the low magnification negatives, prints were made to a final magnification of x2,700. Each print contained a length of 40 to 180 /t of the inner endothelial wall of Schlemm's canal (counted in the anteroposterior direction). On these low magnification prints pinocytotic vesicles, for instance, were not visible, but sufficiently large parts of "vacuoles" could be seen with the naked eye as empty spaces within the layer of endothelial cells forming the inner wall of the canal (Fig. 2). All such empty spaces, henceforth called "vacuoles/' down to about 0.5 mm. diameter were counted, as were all profiles of nuclei, henceforth called "nuclei/ 1 in the same endothelial layer. The "nuclei" were more difficult to identify at low magnification because of lower contrast and the threshold size for detection was 4 to 5 times larger. Since the number of "vacuoles" and "nuclei" were to be expressed per unit length of the endothelial wall, this length was measured along the endothelial surface and rounded off to the nearest 5 p. Statistical treatment. Since the different sections from each animal were not of equal length, the total number of "vacuoles" or "nuclei" was counted in the five sections and divided by the total length of the five sections. This figure is an average characterizing that eye. In estimating significances, individual differences or ratios were formed for each animal and these counted as statistical units. Results Table I shows the pilocarpine effect on facility and resistance and the total volume of perfusion fluid, which entered the anterior chambers during the measurement. The Fig. 2. Electron micrograph of the area marked in Fig. 1 with the same labeling. V is the "vacuole" in the endothelium of the inner wall of Schlemm's canal. (x2,700.)

4 56 llohnberg and Bdrdny Investigative Ophthalmology February I960 Table I. Effects of 200 /*g pilocarpine HCl, put onto cornea one hour before the experiment Animal 1,114 1,115 1,116 1,117 1,118 L,119 1,120 Average 1,113 Facility (lil/mm. Hg/min.) Experimental Control Control eye Resistance difference (mm. Hg/ lil min.-') needle blocked Total volume of infused fin id. ( Experimental \ Control Perfusion (min.) Table II. Electron-microscopic changes one hour after pilocarpine HCl, 200 jug put onto cornea. Each animal counted as a statistical unit Animal 1,113 1,114 1,115 1,116 1,117 1,118 1,119 1,120 Average Average difference Vacuoles per 1,000 n Experimental \ Control ± < P ± < 0.02 Nuclei per 1,000 n Experimental \ Control ±6.9 ± ±5.9 n. s. Total length of sections Experimental \ Control Average ratio Experimental/ control P < ±0.15 n. s. volume is larger on the experimental side but still is only a moderate fraction of the anterior chamber volume which is about 250 fx\ in these monkeys. The drug evidently had caused a good reduction in resistance in all cases. Electron microscope findings for these animals are summarized in Table II. To our intense surprise (the eyes were coded and the electron microscopist did not know which was control and which was experimental) there was an unquestionable decrease in the frequency of "vacuoles" in the experimental eyes. There was no corresponding change in the frequency of the reference structures, the "nuclei." The surprising nature of this finding caused us to look closer at possible errors. First, the question of repeatability of counting, as such, arose. Prints that had been counted several weeks or months earlier were counted once more (without knowl-

5 Volume 5 Number 1 Pilocarpine effect in Schlemm's canal 57 Table III. Repeatability of counting procedure. Prints of 40 control and 40 experimental sections were counted twice Vacuoles, control Vacuoles, experimental Nuclei, control Nuclei, experimental Coefficient of correlation 0.82 n = n = n = n = 40 edge of the previous result) and correlation coefficients between first and second count of each section were calculated. Table III shows the results. Counting is reasonably repeatable. Further, the question of "pseudovacuoles" was checked. It was known from previous studies at high magnifications that there should be rare instances where intercellular spaces could appear as "vacuoles" at low magnification. Also, artifacts of different kinds might appear as empty spaces under low magnification. The same prints as had been counted twice with the naked eye were, therefore, once more gone over with a xlo magnifier. The criterion for acceptance of a "vacuole" now was the typical appearance of the cell membrane around the empty space. The following findings were made: Of 78 "naked eye vacuoles" in experimental eyes, seven were now discarded as membraneless artifacts, but 4 new ones were found. Of 115 "naked eye vacuoles" in control eyes, eight were discarded as membraneless artifacts and three as intercellular spaces, five new ones were found. In the case of "nuclei," which were also checked, there were no changes. Thus, the error caused by counting at low magnification was not serious. Moreover, some of the membraneless empty spaces may have been real vacuoles with the membrane very tangentially cut. We, therefore, feel justified in stating that the differences shown in Table II represent differences in the number of real sections through real vacuoles, perforating channels or blind-ending imaginations. We have no morphological criteria to distinguish between these three possibilities on individual sections. Different correlations were then tested. There was no correlation between the number of "vacuoles" and "nuclei," either in the series of experimental eyes or in that of the controls. Nor was there any correlation between pretreatment or posttreatment facility and "vacuole" count, or between the drug-induced resistance decrease or facility increase and the change in "vacuole" count. It seems improbable, therefore, that the change in "vacuole" count is causal with respect to the resistance change observed after pilocarpine. There is also no correlation between the amount of perfusion fluid which had entered the anterior chamber and the "vacuole" count, either if absolute values are used or if the two variables are related to those of the control side. Discussion Pilocarpine treatment undoubtedly results in strong contraction of the ciliary muscle. This could possibly cause mechanical stretching of the endothelial wall in the anteroposterior direction and reduce the number of counted structures per unit length in this direction. The counts of "nuclei" were done to control this point. The fact that the average count in the experimental eyes did not differ significantly from that in the corresponding control eyes shows that simple surface increase of the endothelial cells must play a very minor role in the observed effect. In fact, the reduction in the number of structures counted per unit length can equally well be due to a change in their shape or true size as in their true frequency per unit of endothelial surface. The sections cut for electron microscopy are so thin compared with the size of the structures in question that the statistical chance of being included in a section is directly proportional to the size (in the relevant direction) of the structure. If, under the action of pilocarpine, channels through cells were to straighten out and shorten, they

6 58 Holmberg and Bdrdnij Iiwcstigatioe Opluhulmology February 1966 could, at the same time, offer less resistance to fluid outflow and stand less of a chance to become included in a section. However, the observed reduction in "vacuole" count could also be due to a decrease in size or change in shape or true vacuoles or of blind imaginations. Changes could be due to passive stretching of the endothelium or to a direct action of pilocarpine. Clearly the matter needs further study. REFERENCES 1. Barany, E. H.: The mode of action of pilocarpine on outflow resistance in the eye of a primate (Cercopithecus aethiops), INVEST. OPHTII. 1: 712, llolmberg, A.: The fine structure of the inner wall of Schleinm's canal, Arch. Ophth. 62: 956, I-folmberg, A.: Schlemm's canal and the trabecular meshwork. An electron microscopic study of the normal structure in man and monkey (Cercopithecus aethiops), Docum. Ophth! J9: 339, Barany, E. H.: Applanation tonometry and ophthalmoscopy of the vervet monkey (Cercopithecus aethiops) in phencyclidine catalepsia, INVEST. OPHTH. 2: 322, Tornqvist, C: Comparative studies of the effect of pilocarpine on the pupil and on the refraction in two species of monkey (Cercopithecus aethiops and Macaco irus), INVEST. OPHTH. 3: 388, Sears, M. L.: Miosis and intraocular pressure changes during manometry in mechanically irritated rabbit eyes studied with improved manometric technique, Arch. Ophth. 63: 707, Barany, E. H.: Simultaneous measurement of changing intraocular pressure and outflow facility in the vervet monkey by constant pressure infusion, INVEST. OPHTH. 3: 135, Zetterqvist, H.: The ultrastructural organization of the columnar absorbing cells of the mouse jejunum, Dissertation, Karolinska Inst., Stockholm, Luft, J. H.: Improvements in epoxy resin embedding methods,. Biophys. & Biochem. Cytol. 9: 409, 1961.